Integrin and growth factor signaling pathways regulate multiple cellular behaviors including cellular migration for various biological events such as animal development and survival. Abl family non-receptor tyrosine kinases, and Crk and Nck families of Src Homology 2 (SH2)/SH3 adaptor proteins are known to regulate actin cytoskeleton rearrangement and focal adhesion turnover to modulate cellular migration. However, the functional interactions among these three families of proteins remain elusive in these two important signaling pathways. ^ In a 1st part of my thesis project, I showed that the c-Abl PxxP motifs, which bind SH3 domains, are indispensable for the coordinated regulation of filopodium and focal adhesion formation and cell spreading dynamics during cell attachment. Candidate Abl PxxP motif binding partners were identified by screening a comprehensive SH3 domain phage display library. A combination of protein overexpression, silencing, pharmacological manipulation and mutational analysis demonstrated that the PxxP motifs of c-Abl exert their effects on actin organization by two distinct mechanisms, involving the inhibition of Crk signaling and the engagement of Nck. ^ In a 2nd part, I studied the roles of Crk family adaptors in platelet-growth factor receptor β (PDGFβR) signaling using siRNA-based gene silencing combined with mutational analysis. I showed that Crk family adaptors are essential for coordination of actin cytoskeleton rearrangement and focal adhesion turnover to promote PDGF-mediated cell migration. Interestingly, Crk adaptors were required only during the early period of PDGFβR signaling, and inhibited in large part by Abl family tyrosine kinases shortly after PDGF stimulation. Our data also suggest that Crk adaptors regulate cytoskeletal rearrangements through the selective activation of the small GTPases Rac1 or Rap1, and that the C-terminal SH3 domain of Crk determines which GTPase is activated. ^ My studies revealed previously unappreciated roles of c-Abl PxxP motifs in regulation of cell spreading and essential roles of Crk adaptors in PDGFβR signaling. The new insights gained from my thesis project provided molecular details for how these proteins work together to orchestrate actin cytoskeleton rearrangement and focal adhesion dynamics. These will help to elucidate developmental and pathological events in where these proteins play critical roles.
